Description
Recognition site and hydrolysis position:
| GC(5mC)GGC | G↑C(5mC)GGC |
| CGG(5mC)CG | CGG(5mC)C↓G |
Source: Kocurea rosea 307
Substrate specificity:
Unit definition: One unit is defined as the amount of enzyme required to hydrolyze completely 1 μg of linearized plasmid pMHpaII 1 in 1 hour at 37°C in a total reaction volume of 50 μl.
| KroI activity assay on DNA pMHpaII 1/DriI Lanes: 1 and 6 – 1 Kb SE DNA-markers. 2 – Control DNA pMHpaII 1/DriI, 3 – 0.5 μl Kro I 4 – 1 μl Kro I 5 – 2 μl Kro I Products were separated in 1,4% agarose gel in Buffer TAE. |
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Assayed on: DNA pMHpaII 1/DriI is a linearized plasmid pMHpaII 1. pMHpaII 1 carries a gene of DNA-methyltransferase M.HpaII, which methylates sites 5`-CCGG-3` producing 5`-C(5mC)GG-3`/3`-GG(5mC)C-5`, and includes three canonical sites
5`-GC(5mC)GGC-3`/3`-CGG(5mC)CG-5`.
Optimal SE-buffer: SE-buffer G
Enzyme activity (%):
| B | G | O | W | Y | ROSE |
| 50 | 100 | 25 | 50 | 75 | 100 |
Storage conditions: 10 mM Tris-HCl (pH 7.5); 200 mM NaCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 200 μg /ml BSA; 50% glycerol; Store at -20°C.
Ligation: –
Non-specific hydrolisis: No detectable degradation of 1μg of Lambda DNA was observed after incubation with 1 units of enzyme for 16 hours at 30°C in a total reaction volume of 50 μl.Reagents Supplied with Enzyme
10 X SE-buffer G
Methylation sensitivity: The enzyme cleaves C5-methylated DNA and doesn’t cut unmodified DNA [1].
Notes: References:
1. Dedkov V.S., Rechkunova N.I., Degtyarev S.Kh., Zhilkin P.A., Kolykhalov A.A., Verkhozina V.A. Bacillus sphaericus – a strain producer of restriction endonuclease BsiI.// Soviet Union patent SU 1784642 (1992). (In Russian)
2. Chernukhin V.A., Zhuravleva R.O., Tarasova G.V., Boltengagen A. A., Akishev A.G., Mikhnenkova N.A., Degtyarev S.Kh. Bacterial strain Kocuria rosea – producer of KroI site specific endonuclease. // Russian Federation patent RU 2394099 C1 (2010).
1. V.A. Chernukhin, E.V. Kileva, J.E. Tomilova, A.A. Boltengagen, V.S. Dedkov, N.A. Mikhnenkova, D.A. Gonchar, L.N. Golikova, S.Kh. Degtyarev A new methyl-directed site-specific endonuclease KroI recognizes and cuts DNA sequence 5’-G^C(5mC)GGC-3’ // Translated from “Ovchinnikov bulletin of biotechnology and physical and chemical biology” V.7, No 1, pp 14-20, 2011
2. Chernukhin V.A., Zhuravleva R.O., Tarasova G.V., Boltengagen A. A., Akishev A.G., Mikhnenkova N.A., Degtyarev S.Kh. Bacterial strain Kocuria rosea – producer of KroI site specific endonuclease. // Russian Federation patent RU 2394099 C1 (2010).
